Lipopeptides and their use in the treatment of mastitis in mice

Abstract

The present application provides a kind of lipopeptide, the sequence of the lipopeptide is: C18-Lys-Gly-Gly-Lys-NH2.The present application also provides the application of the lipopeptide in treating mouse mastitis.The lipopeptide of the present application is simple in design, low in preparation cost, compared with traditional antibiotics, the unique antibacterial mode of lipopeptide makes it difficult for bacteria to develop resistance through conventional mutation, and the risk of drug resistance is low after long-term use, which can maintain the therapeutic effect for a longer period.The C18KGGK lipopeptide of the present application can significantly inhibit the malignant progression of inflammatory sites, cause little harm to the body, inhibit the generation of inflammatory factors, and promote the healing and recovery of inflammatory cells.Therefore, the lipopeptide of the present application has good application prospect in the preparation of clinical antibacterial drugs, and has the potential to become a new type of antibiotic candidate drug.

Description

Technical Field

[0001] This invention relates to lipopeptides and their application in the treatment of mastitis in mice. Background Technology

[0002] Mastitis is mainly caused by pathogenic microorganisms such as *Streptococcus agalactiae* invading the mammary ducts. Clinical manifestations include mammary redness and swelling, abnormal milk production, and a sudden drop in milk yield. In severe cases, it can lead to mammary fibrosis and even systemic sepsis. In mouse research models, mastitis not only affects the health and reproductive performance of mice but also seriously hinders related biomedical research. For farmed mice used in scientific research and biopharmaceutical production, the prevention and treatment of mouse mastitis is crucial. Currently, antibiotics are the common treatment for mouse mastitis. However, recurrent mastitis caused by persistent pathogens can lead to antibiotic resistance. This not only makes further medication use difficult for veterinarians but also poses a potential threat to public health. Therefore, reducing or discontinuing the use of antibiotics is an inevitable trend in the prevention and treatment of mastitis. Traditional Chinese medicine treatments are currently in the exploratory stage and face problems such as long treatment cycles, unstable efficacy, and unclear effective substances. Therefore, developing a safe and effective new method or product for treating mastitis is of significant practical importance.

[0003] In recent years, lipopeptides have attracted much attention due to their novel mode of action and broad-spectrum antibacterial activity against a variety of pathogens. Traditional antibiotics mainly kill bacteria by inhibiting bacterial cell wall synthesis or interfering with bacterial protein or nucleic acid synthesis, which may lead to selective resistance in bacteria. However, because lipopeptides have lipophilic activity, they can kill bacteria by disrupting their cell membranes, causing irreparable damage. Therefore, bacteria are less likely to develop resistance to lipopeptides compared to traditional antibiotics. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides lipopeptides and their application in the treatment of mastitis in mice.

[0005] The first objective of this invention is to provide a lipopeptide with the sequence: C18-Lys-Gly-Gly-Lys-NH2; the structural formula of the lipopeptide is shown in formula (Ⅰ) below:

[0006]

[0007] (I).

[0008] A second objective of this invention is to provide the use of the above-mentioned lipopeptide in the preparation of a medicament for treating mastitis in mice.

[0009] Preferably, the mastitis is caused by infection with Streptococcus agalactiae.

[0010] Preferably, the drug is an injectable form.

[0011] Preferably, the concentration of lipopeptides in the drug is 1 mg / mL to 10 mg / mL.

[0012] A third objective of this invention is to provide a medicament in which the active ingredient comprises the aforementioned lipopeptide.

[0013] Preferably, the drug is an injectable form.

[0014] Preferably, the concentration of lipopeptides in the drug is 1 mg / mL to 10 mg / mL.

[0015] The lipopeptides of this invention are simple in design and inexpensive to prepare. Compared with traditional antibiotics, the unique antibacterial mechanism of lipopeptides makes it difficult for bacteria to develop drug resistance through conventional mutations, resulting in a lower risk of drug resistance after long-term use and a more sustained therapeutic effect. The C18KGGK lipopeptide of this invention can significantly inhibit the malignant progression of inflammation sites, cause minimal harm to the body, inhibit the production of inflammatory factors, and promote the healing and recovery of inflammatory cells. Therefore, the lipopeptides of this invention have excellent application prospects in the preparation of clinical antibacterial drugs and are expected to become candidate drugs for novel antibiotics. Attached Figure Description

[0016] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0017] Figure 1 This is a graph showing the hemolytic activity of the lipopeptide of the present invention.

[0018] Figure 2 This is a diagram illustrating the therapeutic effect of the lipopeptide of the present invention on mastitis in mice.

[0019] Figure 3 A comparative diagram showing the therapeutic effects of different lipopeptides on mastitis in mice.

[0020] Figure 4 HE staining results of mammary gland tissue sections from C18KGGK mice.

[0021] Figure 5 HE staining results of mammary tissue sections from mice in different lipopeptide treatment groups.

[0022] Figure 6 The results are statistical analysis of the bacterial load assay in mouse mammary tissue.

[0023] Figure 7 The results of statistical analysis of IL-6 and TNF-α factors in the detection of inflammatory factors in mouse mammary tissue. Detailed Implementation

[0024] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0025] Example 1: Preparation of lipopeptides

[0026] The three lipopeptides in this embodiment are fatty acid-modified polypeptides containing two or four amino acids. They are obtained by combining lysine (Lys, K) with each other or with glycine (Gly, G) to obtain ultrashort-sequence polypeptides containing two or four amino acids. Then, the C-terminus of these ultrashort-sequence polypeptides is amidated, and the N-terminus is modified with C16 or C18 saturated straight-chain fatty acids, resulting in the following structural formulas: C16-Lys-Gly-Gly-Lys-NH2, labeled C16KGGK; C18-Lys-Lys-NH2, labeled C18KK; C18-Lys-Gly-Gly-Lys-NH2, labeled C18KGGK. The above lipopeptides were prepared using a classic solid-phase synthesis method by Shanghai Qiangyao Biotechnology Co., Ltd. (Shanghai, China), and the products are in lyophilized powder form. The lipopeptides were prepared into a stock solution with a final concentration of 10 mg / mL using sterile PBS and stored at -80°C for later use.

[0027] The structural formula of C18KGGK is as follows:

[0028]

[0029] Example 2: Determination of Minimum Inhibitory Concentration (MIC)

[0030] The minimum inhibitory concentration (MIC) of three lipopeptides was determined using the microbroth dilution method to evaluate the antimicrobial activity of novel derived peptides. The tested Streptococcus agalactiae GS032 strain was cultured in tryptone soybean broth (TSB) at 37°C to mid-log phase, and then diluted to 5 × 10⁻⁶. 5 CFU / mL. The peptide solutions were diluted twofold to final concentrations ranging from 0.05 to 400 μg / mL. 10 μL of the peptide solution and 90 μL of logarithmic-phase bacterial suspension were added to 96-well microtiter plates and incubated at 37°C for 18–24 hours. All assays were repeated three times. The minimum inhibitory concentration (MIC) was defined as the lowest concentration at which no visible bacterial growth was observed after overnight incubation. The results are shown in Table 1. By measuring the MIC, it was found that all three lipopeptides exhibited some antibacterial activity against *Streptococcus agalactiae*.

[0031] Table 1 Minimum inhibitory concentrations of synthetic lipopeptides

[0032] GS032 3.125 3.125-6.25 3.125-6.25

[0033] Example 3: Red Blood Cell Hemolysis Experiment

[0034] Mouse blood cells were collected and washed three times with 0.9% NaCl solution. 100 μL of 2% (v / v) erythrocyte solution was mixed with 100 μL of peptides (C16KGGK, C18KK, and C18KGGK) to achieve a final peptide concentration of 0.2–400 μg / mL. After incubation at 37°C for 1 hour, the mixture was centrifuged at 1000 rpm / min for 10 minutes at 4°C. 100 μL of the supernatant was transferred to a new microcentrifuge tube, and the absorbance (ODS) was measured at 540 nm. The values ​​of 0.9% NaCl solution (ODB) and 0.1% Triton X-100 solution (ODP) were used as controls. The percentage of hemolysis of the peptides was calculated using the following formula: Hemolysis rate (%) = [(ODS–ODB) / (ODP–ODB)] × 100%.

[0035] The results are as follows Figure 1 As shown, the hemolysis assay results indicate that all three lipopeptides exhibit hemolytic activity at high concentrations. When the concentration of C16KGGK is below 12.5 μg / mL, over 93% of erythrocytes remain intact. C18KK at 12.5 μg / mL causes approximately 15% hemolysis, and C18KGGK at 100 μg / mL also causes approximately 15% hemolysis. C18KGGK exhibits extremely low hemolytic activity. Taking the concentration of the lipopeptide that maintains 80% erythrocyte integrity as its maximum concentration, the safe erythrocyte concentrations for C16KGGK, C18KK, and C18KGGK are 12.5 μg / mL, 12.5 μg / mL, and 100 μg / mL, respectively. The hemolytic activity of lipopeptides is the most commonly used method for preliminary assessment of the safety of lipopeptide drugs. Hemolytic activity can be used as an indicator of drug safety. By comparison, the hemolytic activity of C18KGGK is significantly lower than that of C16KGGK and C18KK, indicating higher safety.

[0036] Figure 1 This is a graph showing the hemolytic activity of the lipopeptide of the present invention.

[0037] Example 4: Establishment of a mouse mastitis model

[0038] 1. For primiparous, lactating female mice 4-6 days postpartum, after inhalation anesthesia, place them in a supine position, and disinfect the abdominal mammary glands and surrounding skin with 75% alcohol. Infuse non-lactate streptococci GS032 into each mammary gland using a microsyringe via the mammary ducts, injecting 5 × 10⁻⁶ ml per mammary gland. 5 An infection model of strain GS032 was constructed using CFU / mL resuspension of Streptococcus agalactiae.

[0039] 2. Lipopeptide administration began 24 hours after injection of *Streptococcus agalactiae*. Before lipopeptide treatment, the mother mouse and pups were separated for 2 hours. After the mammary glands were full of milk, the mother mouse was anesthetized, and 10 μg of the lipopeptide solution prepared in Example 1 (concentration 1 mg / mL) was injected through the mammary duct. Two hours after injection, the mother mouse and pups were placed in the same cage. The medication was administered once daily for 7 days, followed by a 2-day rest period after the treatment trial. The mice's condition was observed daily.

[0040] 3. The experiment was divided into 5 groups, with 3 mice in each group. The experimental groups are as follows:

[0041] (1) N group (control group): Mice were not treated in any way.

[0042] (2) P group (mastitis group): a mouse mastitis model caused by Streptococcus non-lactaria, without any treatment.

[0043] (3) Lipopeptide treatment group: a mouse mastitis model infected with Streptococcus agalactiae was treated by injecting C16KGGK lipopeptide prepared in Example 1 into the mammary duct.

[0044] (4) Lipopeptide treatment group 2: a mouse mastitis model infected with Streptococcus agalactiae, treated by injection of C18KK lipopeptide prepared in Example 1 into the mammary duct.

[0045] (5) Lipopeptide treatment in three groups: a mouse mastitis model infected with Streptococcus agalactiae, treated by injection of C18KGGK lipopeptide prepared in Example 1 into the mammary duct.

[0046] 4. Results are as follows Figure 2 , Figure 3 As shown, mice in group N had normal mammary glands and did not have mastitis; mice in group P had mastitis; and mice in the C18KGGK group experienced reduced mastitis symptoms after treatment. This indicates that the C18KGGK lipopeptide of this invention has a certain inhibitory effect on mastitis during injection treatment and can promote the healing time of a mouse mastitis model without Streptococcus lactis infection. Comparing the therapeutic effects of the three lipopeptides, the mastitis symptoms in all treatment groups were reduced, while some areas of the mammary gland in the C18KK treatment group still showed inflammation, congestion, or bleeding.

[0047] Figure 2 This is a diagram illustrating the therapeutic effect of the lipopeptide of the present invention on mastitis in mice.

[0048] Figure 3 A comparative diagram showing the therapeutic effects of different lipopeptides on mastitis in mice.

[0049] Example 5: Pathological section analysis of breast tissue

[0050] Pathological analysis was performed on the mouse mastitis model constructed in Example 4. The specific experimental steps are as follows:

[0051] 1. Dewaxing paraffin sections to water: Place the sections in xylene I (20 min), xylene II (20 min), anhydrous ethanol I (10 min), anhydrous ethanol II (10 min), 95% ethanol (5 min), 90% ethanol (5 min), 80% ethanol (5 min), 70% ethanol (5 min), and wash with distilled water (10 s);

[0052] 2. Staining: Immerse the sections in hematoxylin solution for 5 minutes, rinse with tap water, differentiate and rinse with 1% hydrochloric acid alcohol for 5 seconds, and stop when the sections turn light red. Place them under running water to restore the blue color, and then stain with eosin for 15 minutes.

[0053] 3. Dehydration and mounting: Place the sections in 95% alcohol I (5 min), 95% alcohol II (5 min), anhydrous ethanol I (5 min), anhydrous ethanol II (5 min), xylene I (5 min), and xylene II (5 min) in sequence to dehydrate and clear them. Remove the sections from the xylene and let them dry slightly. Mount them with neutral resin and observe and photograph them under a microscope.

[0054] The results are as follows Figure 4 , Figure 5 As shown, no obvious inflammatory symptoms were observed in mammary gland sections of mice in group N; mice in group P showed obvious inflammatory symptoms in their mammary glands, with abundant serous fluid and inflammatory cells within the mammary alveoli, and incomplete alveolar structure; mice in the C18KGGK treatment group had very few inflammatory cells in their mammary alveoli, with clear boundaries, relatively intact structure, and no fibrosis in the stroma. Mice in the C16KGGK and C18KK treatment groups showed reduced inflammatory cells in their mammary alveoli, and relatively intact alveolar structure.

[0055] Figure 4 HE staining results of mammary gland tissue sections from C18KGGK mice.

[0056] Figure 5 HE staining results of mammary tissue sections from mice in different lipopeptide treatment groups.

[0057] Example 6: Determination of bacterial load in breast tissue

[0058] Bacterial load was determined in the mouse mastitis model constructed in Example 4. The specific experimental steps are as follows:

[0059] 0.1 g of mammary gland tissue was weighed from laboratory mice and homogenized in 0.9 mL of sterile PBS solution. The tissue homogenate was serially diluted, and 100 μL of each dilution was spread onto TSB plates. The inoculated culture dishes were inverted and incubated overnight at 37°C. The colonies growing on the plates were counted and statistically analyzed.

[0060] The results are as follows Figure 6As shown, the number of bacteria in the mammary tissue of C18KGGK mice was significantly lower than that in untreated mastitis mice.

[0061] Figure 6 The results are statistical analysis of the bacterial load assay in mouse mammary tissue.

[0062] Example 7: Measurement of Inflammatory Factors in Breast Tissue

[0063] Inflammatory factors were measured in the mouse mastitis model constructed in Example 4. The specific experimental steps are as follows:

[0064] The expression of inflammatory factors in mammary gland tissue was detected using real-time quantitative polymerase chain reaction (PCR). Total RNA was extracted from mouse mammary gland tissue using TRIzol reagent according to the manufacturer's instructions. cDNA was synthesized by reverse transcription using PrimeScript™ reverse transcriptase mixture according to the manufacturer's instructions. GreenMaster Mix (High ROX Premixed) was used to detect the expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in breast tissue using quantitative real-time PCR (qRT-PCR) to determine the degree of inflammation. Primer sequences are shown in Table 2. The reaction system was as follows: qPCR 10 μL Green Master Mix; 0.4 μL reverse primer; 0.4 μL forward primer; 5 μL cDNA; 4.2 μL double-distilled water. Reaction conditions were as follows: pre-denaturation, 95 °C, 30 sec; 40 cycles, 95 °C for 10 sec, 60 °C for 30 sec. Results were calculated using the comparative Ct method (2-ΔΔct) and normalized to the internal control level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0065] Table 2 Primer sequences for real-time quantitative PCR

[0066]

[0067] The results are as follows Figure 7 As shown, compared with the mammary tissue of the P group mice, the expression levels of IL-6 and TNF-α in the mammary tissue of the C18KGGK group mice were significantly reduced.

[0068] Figure 7 The results of statistical analysis of IL-6 and TNF-α factors in the detection of inflammatory factors in mouse mammary tissue.

[0069] Figures 1-7 In the text, * indicates p < 0.01, and **** indicates... <p<0.0001。

[0070] The above embodiments confirm the effectiveness of the C18KGGK lipopeptide prepared in this invention. Through minimum inhibitory concentration (MIC) assays and erythrocyte hemolysis experiments, it was found that C18KGGK exhibits certain antibacterial activity against *Streptococcus agalactiae*, and its hemolytic activity is significantly lower than that of C16KGGK and C18KK, indicating higher safety. Through the construction of a mouse mastitis model, lipopeptide injection treatment, and pathological section observation, it was found that C18KGGK lipopeptide can promote the healing of inflamed tissues, with a better therapeutic effect than C16KGGK and C18KK. Bacterial load assays and inflammatory cytokine assays confirmed that C18KGGK lipopeptide has an inhibitory effect on inflammatory factors, thereby promoting the recovery of inflamed tissues.

[0071] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of lipopeptides in the preparation of drugs for treating mastitis in mice, characterized in that: The sequence of the lipopeptide is: C18-Lys-Gly-Gly-Lys-NH2; the structural formula of the lipopeptide is shown in formula (Ⅰ) below: ； The mastitis was caused by infection with Streptococcus agalactiae.

2. The application according to claim 1, characterized in that: The drug is an injectable form.

3. The application according to claim 2, characterized in that: The concentration of lipopeptides in the drug is 1 mg / mL to 10 mg / mL.

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